Substances KF-1040 and process for producing of the same

ABSTRACT

A process for producing a substance KF-1040A represented by formula (I), and another substance KF-1040B represented by formula (II), which comprises culturing microorganisms capable of producing the substances KF-1040A and KF-1040B, thus accumulating these KF-1040A and/or KF-1040B in the liquid culture and harvesting the same from the culture. Because of the activities of inhibiting diacyloglycerol transferase and sphingomyelinase, the above substances are useful in preventing and treating arteriosclerosis, obesity, thrombus, inflammation and immune function-related diseases.

This application is a 371 of PCT/JP98/00617 filed Feb. 19, 1998.

FIELD OF THE INVENTION

The present invention relates to novel KF-1040 substance having aninhibitory activity for lipid metabolism as well as to a process forproducing such substance.

PRIOR ARTS

There have been known anti-obesity drugs and drugs for hyperlipidemia.For example, centrally acting anorectics will suppress appetite, whichmay, however, be harmful for health due to reduction of appetite.Therefore, it has been expected to develop a new anti-obesity drug or atherapeutic drug for hyperlipidemia which reveals no side effect.

On the other hand, it has recently been made clear that hydrolysis ofsphingomyelin which is one of lipids constituting a biomembrane hasinvolved in the intracellular signal transduction by cytokine, such asinterleukin 1β or tumor necrosis factor α [Y. A. Hannun, J. Biol. Chem.,269, 3125-3128 (1994) and R. Kolesnick & D. W. Golde, Cell, 77, 325-328(1994)], and in the intracellular signal transduction upon activation ofT cells [L. M. Boucher et al, J. Exp. Med., 18, 2059-2068 (1995) and A.Ochi, Medicinal Immunol., 28, 397-401 (1994)] and has a function in thediseases such as arteriosclerosis, inflammations, thrombosis and so onand in the immunoregulation mechanisms therefor. However, anyprophylactic or therapeutic medicament for these diseases has not yetbeen developed in practice from a standpoint of the specific andpotential inhibit or for sphingomyelinase, a hydrolase of sphingomyelin.

Problem to be Solved by the Invention

In recent years, increase in the population of patients with life-stylerelated diseases has brought about large problems in the therapeutic andpreventive medical sciences. Especially, diseases of obesity andhyperlipidemia by accumulation of triacylglycerols due to recent habitof luxurious diet may often lead to more serious diseases such asarteriosclerosis, fatty liver, hypertension, diabetes, coronary heartdisease, stroke, gallbladder disease, osteoarthritis, respiratoryproblems and some types of cancer.

Obesity refers to a physical state in which the stored fat, constitutedmainly of triglycerides, is accumulated excessively in the body,ascribed to an increased synthesis of triacylglycerols causingextraneous accumulation of fat in the adipose tissue. Alsotriacylglycerolaemia is believed to be triggered by facilitation oftriacylglycerol synthesis in intestine and in liver causing, thus, alipoproteineamia with a high concentration of triacylglycerols in blood.Therefore, it is assumed that any substance exhibiting an inhibitoryaction onto diacylglycerol:acyltransferase which involves the selectivesynthesis of triacylglycerols may have an ability for suppressingaccumulation of triacylglycerols in adipose tissue and blood and may beeffective for the therapy of these diseases.

Under the circumstances, it is believed to be worthwhile in the therapyof obesity and hyperlipidemia and of degenerative diseases, such asarteriosclerosis and so on, originated thererfrom, to provide asubstance having an activity of inhibitingdiacylglycerol:acyltransferase.

Furthermore, it is also expected that a substance having an activity ofinhibiting sphingomyelinase which causes hydrolysis of sphingomyelin, abiomembrane constituting lipid, may be useful as a drug foranti-arteriosclerosis, antithrombosis and antiinflammation and as animmunosuppresant, based on a novel functional mechanism not foundheretofore.

Means for Solving Problem

The inventors had conducted researches for metabolic products producedby microorganisms and found that substances which have activities forinhibiting diacylglycerol:acyltransferase and sphingomyelinase wereproduced in the culture medium on the cultivation of a newly identifiedfungal strain KF-1040 isolated among sea weed. These active substancescapable of inhibiting metabolism of lipids were then isolated from theabove-mentioned culture medium and purified, wherefrom the chemicalstructures thereof were determined as represented by the formulae (I)and (II) given below. Since the substances represented by these formulae(I) and (II) were not known in the past, the inventors have named themas “KF-1040 substance A” and “KF-1040 substance B”, respectively, whichare referred to totally as the “KF-1040 substance”.

The present invention has been completed based on the knowledges givenabove and it relates to the KF-1040 substance comprising the KF-1040substance A represented by the following formula (I), namely,

and the KF-1040 substance B represented by the following formula (II),namely,

The present invention further relates to a process for producing novelKF-1040 substance comprising culturing a microorganism which belongs tothe genus Gliocladium and has an ability of producing KF-1040 substanceA and/or KF-1040 substance B in a culture medium, causing to accumulatethe resulting KF-1040 substance A and/or KF-1040 substance B in theculture medium and isolating the KF-1040 substance A and/or KF-1040substance B from the culture medium.

The present invention also relates to a process for producing theKF-1040 substance, wherein the microorganism which belongs to the genusGliocladium and has an ability of producing KF-1040 substance A and/orKF-1040 substance B is Gliocladium sp. KF-1040(FERM BP-6251). Thepresent invention further relates to a microorganism which belongs tothe genus Gliocladium and has an ability of producing KF-1040 substanceA and/or KF-1040 substance B.

The microorganism having the ability for producing the KF-1040 substancerepresented by the formulae (I) and (II)(referred to hereinafter as“KF-1040 material producing fungus”) belongs to the genus Gliocladiumand, for example, the fungal strain Gliocladium sp. KF-1040 isolated bythe inventors is an example to be utilized at the most effectivelyaccording to the present invention. The taxonomical properties of thisproducing strain KF-1040 are as given below:

1. Morphological Properties

This strain grows relatively good in media containing 50% of seawater(with salt concentration of 3.4%), such as potato glucose agar, cornmealagar, malt extract agar, Miura agar medium and seawater starch agar,with abundance of conidia.

On microscopic observation of colony grown on a cornmeal agar medium,the hypha is transparent and has a septum. The conidiophore assumes bothpenicillate and verticillate forms. The penicillate conidiophore (havinga length of 100-200 μm) erects or branches from the basal hypha andforms at the top end or at the branch several penicillate cyclicphialides of sizes of 2.5-3.0 μm×10-23μm, on which a conidial mass isformed.

On the other hand, the verticillate conidiophore (having a length of25-50 μm) erects from the basal hypha and forms at the top end or at thebranch phialides (of sizes of 3.0-5.0 μm×17-25 μm) of a form of elongateflask or of cone converging toward the top, from which a sole conidialmass is formed. The conidium is colorless and has a shape of ellipsoidor elongate ellipsoid of a size of 2.5-3.0 μm×3.0-5.0 μm, rarely withsharp tip at its one end, for the penicillate conidiophore. For theverticillate, the conidium is colorless and has a ellipsoidal orelongate ellipsoidal form of a size 2.5-3.0 μm×6.0-8.5 μm

2. Cultured Properties on Various Media

The results of visual observation of the state of culture of this strainin various culture media at 25° C. for 14 days were as given in thefollowing Table 1.

TABLE 1 Growth condition on the medium Color of Color of (diameter ofsurface of reverse side Soluble Medium colony) colony of colony pigmentPotate-glucose good (28-30 mm) bright gray bright gray none agar mediumfloccose, flat Cornmeal agar good (24-30 mm) bright gray bright graynone medium floccose, stripped Malt extract good (20-22 mm) pale graybright gray none agar medium floccose, flat Miura agar good (22-24 mm)bright gray bright gray none medium floccose, ridged a few Seawaterstarch good (24-27 mm) bright gray white milky none agar mediumfloccose, flat

3. Physiological Properties

(1) Optimum Growth Conditions

Optimum growth conditions of the present strain are: pH 4-8, temperature17-27° C., *seawater concentration 0-50%.

*: salt concentration 3.4% natural seawater is used

(2) Growth Condition

Growth range of the strain is: pH 3-10, temperature 9-32° C., *seawaterconcentration 0-200%

*: salt concentration 3.4% natural seawater is used

(3) Nature:Aerobic

As shown in the above, morphological properties, culture condition andphysiological properties of the present strain KF-1040 as given above,the inventors carried out comparison of this strain with known fungalstrains and reached the identification thereof to be a strain belongingto the genus Gliocladium and named as Gliocladium sp. KF-1040. Thisstrain was deposited on Feb. 6, 1998, at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Science and Technology, under thedeposition No. FERM P-16629, and was then re-deposited on Feb. 12, 1998,at National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Science andTechnology located at 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken.Japan, under the receipt No. FERM BP-6251 from the original depositionby the request for transference to the deposition based on the BudapestTreaty.

As the KF-1040 substance producing fungus to be employed according tothe present invention, while the above-mentioned strain Gliocladium sp.KF-1040 is enumerated, every strain which belongs to the genusGliocladium and produces the KF-1040 substances represented by theformulae (I) and (II) given previously (in the following, referred tointegrally as the “KF-1040 substance” so long as not specificallynoted),including natural mutants, artificial mutants resulting fromirradiation of X-ray and UV ray and by mutational treatment with, suchas N-methyl-N′-nitro-N-nitrosoguanidine and 2-aminopurine, fused strainsand gene-manipulated strains.

On the practical application of the present invention, the KF-1040substance producing fungi belonging to the genus Gliocladium is culturedin a culture medium. As the nutrient sources suitable for producing theKF-1040 substance, nutrient medium containing assimilable carbon sourcesby the microorganism, assimilable nitrogen source by the microorganismand, if necessary, additives including inorganic salts and vitamins areemployed. As the carbon source, saccharides, such as glucose, fructose,maltose, lactose, galactose, dextrin and starch, and vegetable oils,such as soybean oil etc., may be incorporated solely or in combination.As the nitrogen source, there may be employed peptone, yeast extract,meat extract, soybean powder, cotton seed powder, corn steep liquor,malt extract, casein, amino acids, urea, ammonium salts and nitrateseither solely or in combination. Further, salts, such as phosphates,magnesium salts, calcium salts, sodium salts and potassium salts as wellas heavy metal salts, such as iron salts, manganese salts, copper salts,cobalt salts and zinc salts, and vitamins and others adapted to theproduction of the KF-1040 substance may be added adequately.

Upon the culture, antifoaming agent, based on for example, liquidparaffin, animal oil, vegetable oil, silicone and surfactant, may beadded if necessary, when encountered by a severe foaming phenomenon. Thecultivation may favorably be carried out usually in a liquid culturemedium, while liquid and solid media can be employed so long as thenutrient sources given above are contained. In a small scale production,a culture in a flask may be favorable. For producing the objectivematerial industrially in a large amount, an aerating culture withagitation is preferable, as in other fermentation products.

In case the culture is carried out in a large tank, it is preferable topractice in such a manner that the producing fungus is first inoculatedin a relatively small amount of culture medium to cultivate therein, inorder to obviate delay of growth of the fungus, and the culture mixtureis then transferred to the large tank to effect the production culturetherein. Here, it is possible that the culture medium composition is thesame with or different from each other for the pre-culture and for theproduction culture. If necessary, the culture composition may be alteredfor them.

In case the culture is carried out under the condition of aeration withagitation, techniques including mechanical agitation by impellor orother means, rotation or shaking of the fermenter, pumping agitation andair bubbling may be employed in a pertinent manner. The aeration iscarried out under sterilization of air.

The culture temperature may adequately be altered within the range inwhich the KF-1040 substance producing fungus can produce the KF-1040substance, while usually the cultivation is carried out at a temperaturein the range 20-30° C., preferably at around 27° C. The cultivation isperformed usually at a pH of 5-8, preferably at around 7. The durationof cultivation may vary according to each specific culture condition,while 10-20 days are usual.

The KF-1040 substance produced in this manner is present in the thusgrown mycelia and in the cultured filtrate. For purifying the KF-1040substance from the cultured mass, the entire cultured mass is extractedwith a water-missible organic solvent, such as acetone, and the extractis subjected to vaccum evaporation to remove the organic solvent,whereupon the resulting residue is extracted with a water-immisibleorganic solvent, such as ethyl acetate.

In addition to the above-mentioned extraction technique, known practicesemployed for purifying lipid-soluble substances, such as for example,adsorption chromatography, gel filtration chromatography, thin layerchromatography, centrifugal countercurrent distribution chromatography,high performance liquid chromatography, may be employed in a suitablecombination or in repetition to effect separation of the KF-1040substance into each component substance and to purify it.

The physico-chemical properties of the KF-1040 substance A according tothe present invention are as given below:

1) Nature white powder 2) Molecular weight 776 (by fast atom bombardmentmass spectrometry) 3) Molecular formula C₄₀H₇₂O₁₄ 4) Specific rotation[α]_(D) ²⁵ = +62° (c = 0.1, in methanol) 5) UV absorption maximum FIG.1, at 203 nm (ε = 24900), 220 nm (in methanol) (ε = 18000) and 275 nm (ε= 1200) 6) IR absorption maximum FIG. 2, at 1637 cm⁻¹ and 3434 cm⁻¹ (KBrtablet) 7) Proton NMR spectrum as shown in FIG. 3 (in heavy hydrogenmethanol) 8) ¹³C-NMR spectrum as shown in FIG. 4 (in heavy hydrogenmethanol) 9) Solubility in solvents solule in methanol, benzene,chloroform and ethyl acetate; slightly soluble in water and hexane 10)Color reaction positive to sulfuric acid and to phosphorus molybdic acid11) Acidic or alkaline nature neutral

Under the examination of the physico-chemical properties, spectralanalyses data of the KF-1040 substance A as given above, the chemicalstructure of the KF-1040 substance A was determined to be as representedby the following formula (I):

The physico-chemical properties of the KF-1040 substance B according tothe present invention are as given below:

1) Nature white powder 2) Molecular weight 818 (by fast atom bombardmentmass spectrometry) 3) Molecular formula C₄₂H₇₄O₁₅ 4) Specific rotation[α]_(D) ²⁵ = +120° (c = 0.1, in methanol) 5) UV absorption maximum FIG.5 at 204 nm (ε = 43000), 218 nm (in methanol) (ε = 30300) and 272 nm (ε= 2900) 6) IR absorption maximum FIG. 6, at 1633 cm⁻¹ and 3417 cm⁻¹ (KBrtablet) 7) Proton NMR spectrum as shown in FIG. 7 (in heavy hydrogenmethanol) 8) ¹³C-NMR spectrum (in as shown in FIG. 8 heavy hydrogenmethanol) 9) Solubility in solvents soluble in methanol, benzene,chloroform and ethyl acetate; slightly soluble in water and hexane 10)Color reaction positive to sulfuric acid and to phosphorus molybdic acid11) Acidic or alkaline nature neutral

Under the examination of the physico-chemical properties, spectralanalyses data of the KF-1040 substance B as given above, the chemicalstructure of the KF-1040 substance B was determined to be as representedby the following formula (II):

While the description in the above has been directed to the details ofvarious physico-chemical properties of the KF-1040 substance A and ofthe KF-1040 substance B, it is recognized that any compound havingproperties corresponding to the above-identified properties has not beenreported in the literature. Therefore, it is decided that the KF-1040substances are novel substances.

Now, the description is directed to the biological nature of the KF-1040substance A and KF-1040 substance B.

(1) Inhibitory Action to Rat-originated Diacylglycerol:Acyltransferase

The activity of diacylglycerol:acyltransferase was determined by themodified method of Mayorek and Bar-Tana [J. Biol. Chem., 260, 6528-6532(1985)].

Thus, a microsomal fraction prepared from rat liver was used as theenzyme source. To a 175 mM Tris-HCl buffer (pH 8.0) containing 8 mM ofMgCl₂, 1 mg/ml of bovine, serum albumin and 2.5 mM of diisopropylfluorophosphate, there were added 0.75 mM of dioleoylglycerol and 30 μMof [1- ¹⁴C]-palmitoyl-CoA (0.02 μCi) and the total volume was adjustedto 200 μl, whereupon the enzymatic reaction mixture was incubated at 23°C. for 15 minutes. The total lipids were extracted withchloroform/methanol mixture and each lipid was separated by a TLC (withKieselgel GF₂₅₄ and a developer of petroleum ether/diethyl ether/aceticacid of 80/20/1), followed by determination of the radioactivity of thetriacylglycerol fraction using RADIOSCANNER (of the firm AMBIS SystemInc.) to determine the diacylglycerol:acyltransferase activity.

The calculation of the drug concentration corresponding to 50%inhibition of this enzyme gives the values of 16 μg/ml for the KF-1040substance A and 9.0 μg/ml for the KF-1040 substance B.

(2) Inhibitory Action to Formation of Triacylglycerol inHuman-originated Cells (Raji Cell Originated from Human BurkittLymphoma)

The assessment of influence of the substances on the triacylglycerolformation was performed in accordance with the method of Tomoda et al[J. Biol. Chem., 266, 4214-4219 (1991)] using human-originated cells(Raji cell originated from human Burkitt lymphoma).

A Raji cell dispersion of 2.7×10⁶ cells per milliliter containing 0.36nM [1- ¹⁴C]-oleic acid (0.02 μCi) in the presence or absence of the;substance was filled up to a total volume 200 μl, whereupon the reactionwas caused at 37° C. for 30 minutes. The total lipids were extractedwith chloroform/methanol (2/1) mixture. The subsequent procedures werecarried out in the same manner as in the above experiment (1)“inhibitive action to rat-originated diacylglycerol-acyltransferase”.

The calculation of the drug concentration corresponding to 50%inhibition of triacylglycerol formation gives the values of 10 μg/ml forthe KF-1040 substance A and 10 μg/ml for the KF-1040 substance B.

(3) Inhibitory Action to Rat Brain-originated Neutral Sphingomyelilnase

The assessment of influence of the substances on the neutralsphingomyelinase originated from rat brain was performed in accordancewith the modified method of Murakami & Arima [J. Neurochem., 52, 611-618(1989)].

Thus, a membrane fraction prepared from a rat brain was used as theenzyme source and thereto were added 20 mM of HEPES-NaOH buffer solution(pH 7.4), 6.5 mM of MgCl₂, 0.1% Triton X-100 and 25 μM[N-methyl-³H]-sphingomyelin (0.006 μCi) and the mixture was filled up toa total volume of 50 μl. After the reaction at 37° C. for 30 minutes,200 μl of chloroform/methanol mixture (1/2 volume ratio) were added tothe reaction mixture to separate the reaction product[³H]-phosphocholine from the starting material [³H]-sphingomyelin. Thesupernatant layer was taken up into a 50 μl vial. The amount of[³H]-phosphocholine was quantitatively determined by a liquidscintillation counter to estimate the neutrtal sphingomyelinaseactivity.

The calculation of the concentration of the KF-1040 substancecorresponding to 50% inhibition of this enzyme gives the values of 4.2μg/ml for the KF-1040 substance A and 6.1 μg/ml for the KF-1040substance B.

(4) Influence on Human Placenta-Originated Acid Sphingomyelinase

The assessment of influence of the substances on humanplacenta-originated acidic sphingomyelinase was performed in accordancewith the method of Jones et al [Biochem. Journal, 195, 373-382 (1981)]with partial modification.

Thus, an acid sphingomyelinase originated from human placenta (a productof the firm Sigma) was used as the enzyme source and thereto were added250 mM of sodium acetate buffer solution (pH 5.0), 0.1% of NP-40 (of thefirm Sigma), 25 μM [N-methyl-³H]-sphingomyelin (0.006 μCi) and variousconcentrations of the substance, and the mixture was filled up to atotal volume of 50 μl. After the reaction at 37° C. for 30 minutes, 200μl of chloroform/methanol mixture (1/2 volume ratio) was added to thereaction mixture to separate the reaction product [³H]-phosphocholinefrom the starting material [³H]-sphingomyelin. The supernatant layer wastaken up into a 50 μl vial. The amount of [³H]-phosphocholine wasquantitatively determined by a liquid scintillation counter to estimatethe acid sphingomyelinase activity.

The calculation of the concentration of the KF-1040 substancecorresponding to 50% inhibition of this enzyme gives the values of 48μg/ml for the KF-1040 substance A and 24 μg/ml for the KF-1040 substanceB.

As described above, the novel substance according to the presentinvention exhibits activity for inhibitingdiacylglycerol:acyltransferase and sphingomyelinase and, hence, isuseful for the prophylaxis and therapy of patients with diseasesrelating to arterisclerosis, obesity, thrombosis, inflammations andimmunofunctional disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the UV absorption spectrum (in methanol) of the KF-1040substance A according to the present invention.

FIG. 2 shows the IR absorption spectrum (with KBr) of the KF-1040substance A according to the present invention.

FIG. 3 shows the proton NMR spectrum (in heavy methanol) of the KF-1040substance A according to the present invention.

FIG. 4 shows the ¹³C-NMR spectrum (in heavy methanol) of the KF-1040substance A according to the present invention.

FIG. 5 shows the UV absorption spectrum (in methanol) of the KF-1040substance B according to the present invention.

FIG. 6 shows the IR absorption spectrum (with KBr) of the KF-1040substance B according to the present invention.

FIG. 7 shows the proton NMR spectrum (in heavy methanol) of the KF-1040substance B according to the present invention.

FIG. 8 shows the ¹³C-NMR spectrum (in heavy methanol) of the KF-1040substance B according to the present invention.

EXAMPLE

Two 500-ml Erlenmeyer flasks charged each with 100 ml of a liquidculture medium (pH 6.0) prepared by dissolving 2.0% of glucose, 0.5% ofpolypeptone (of Nippon Seiyaku K. K.), 0.2% of yeast extract (ofOriental Kobo Kogyo K. K.), 0.05% of magnesium sulfate 7 hydrate, 0.1%of potassium dihydrogen phosphate and 0.1% of agar in 50% of naturalseawater were inoculated each with one loopful of the strain ofGliocladium sp. KF-1040 (FERM BP-6251) whereupon each inoculated mediumwas cultured at 27° C. for 4 days with shaking.

The resulting cultured medium was used as the cultured seed. 60 Rouxflasks of each 1,000 ml capacity were charged each with 300 ml of aliquid culture medium (pH 6.0) prepared by dissolving 100 g/l of potateand 1.0% of glucose in 50% of natural seawater. After sterilization andcooling, each flask was inoculated aseptically with 3 ml of the cultureseed and the inoculated mixture was cultured at 27° C. for 16 days understanding still.

To the total cultured liquor, 18 litters of acetone were added andagitated well, followed by concentration under a reduced pressure andthe so-concentrated liquor was extracted with ethyl acetate. The extractlayer was subjected to concentration under a reduced pressure, whereby2.2 grams of a crude product were obtained. This crude product wasdissolved in a small amount of acetonitrile and the resulting solutionwas passed to an ODS column (200 g, supplied from Senshu Kagaku K. K.,ODS-SS-1020T) filled with a 30% acetonitrile water. After washing thecolumn with 50% acetonitrile water, the column was eluted with 60%acetonitrile water and, then, with 70% acetonitrile water. From theeluates, 87 mg of a crude product of the substance A and 104 mg of acrude product of the substance B were obtained by concentration underreduced pressure.

Each of the crude products was fractionated by a high performance liquidchromatography (Shiseido Capsulepack, ODS-SG column, 20 mm×250 mm, flowrate=6.0 ml/min.; detection: 215 nm UV using an eluent of 70%acetonitrile water). A fraction eluted at the retention time of 17minute for the substance A and a fraction eluted at the retention timeof 23 minute for the substance B were collected, respectively. Eachfraction was treated by removing the organic solvent and extracting theaqueous layer with ethyl acetate, whereby 16 mg of the KF-1040 substanceA and 40 mg of the KF-1040 substance B were obtained.

EFFECT OF THE INVENTION

As detailed above, the novel KF-1040 substance according to the presentinvention exhibits activity for inhibitingdiacylglycerol:acyltransferase and sphingomyelinase and, hence, isexpected to be useful for the prophylaxis and therapy of patients withdiseases relating to arteriosclerosis, obesity, thromobosis,inflammations and immunofunctional disorder.

What is claimed is:
 1. KF-1040 substance comprising KF-1040 substance Arepresented by the following formula (I), namely,

and KF-1040 substance B represented by the following formula (II),namely,


2. A process for producing KF-1040 substance as defined in claim 1,comprising cultivating a microorganism Gliocladium sp. KF-1040, saidmicroorganism having an ability of producing KF-1040 substance A and/orKF-1040 substance B in a culture medium, causing the resulting KF-1040substance A and/or KF-1040 substance B to accumulate in the culturemedium and harvesting the KF-1040 substance A and/or KF-1040 substance Bfrom the culture medium.
 3. An isolated culture of a microorganismGliocladium sp. KF-1040, said microorganism having an ability ofproducing KF-1040 substance A and/or KF-1040 substance B.
 4. The processfor producing KF-1040 substance according to claim 2, wherein saidculture medium comprises seawater.
 5. The process for producing KF-1040substance according to claim 4, wherein said seawater has a saltconcentration of 3.4%.
 6. The process for producing KF-1040 substanceaccording to claim 2, wherein said culture medium comprises 50% ofseawater.
 7. The process for producing KF-1040 substance according toclaim 2, wherein said microorganism is grown in an aerated and agitatedculture medium.
 8. The process for producing KF-1040 substance accordingto claim 2, wherein said microorganism has been isolated from seaweed.9. The isolated culture of a microorganism according to claim 3, whereinsaid microorganism has been isolated from seaweed.